Genotyping of K-ras exon 2 codons 12 and 13 mutations in colorectal cancer by pyrosequencing

Abstract

Objective To investigate the clinical significance of pyrosequencing assay for determining K-ras mutations in exon 2 codons 12 and 13 in clinical colorectal cancer tissues. Methods Genomic DNA, extracted from K-ras mutant cell lines SW480 (homozygous, c.35G>T), DLD-1 (heterozygous, c.38G>A) and wild-type HT-29, was first used as the sequencing template respectively to test the accuracy of pyrosequencing methodology.The SW480 and DLD-1 DNA was separately mixed with wild-type HT-29 DNA in proportions of 2%, 3%, 5%, 10%, 20%, 30% and 50%, the sensitivity for mutation detection was measured separately by pyrosequencing assay and directed Sanger DNA sequencing in the serial DNA mixture samples.The pyrosequencing assay results were compared with the corresponding Sanger sequencing and the datas were analysized by Fisher exact test.Pyrosequencing analysis was then performed for screening K-ras exon 2 mutations at codons 12 and 13 on DNA isolated from a panel of 30 colorectal cancer samples derived from clinical formalin-fixed and paraffin embedded (FFPE) tissues. Results Cancer cell lines with known K-ras mutations (SW480 and DLD-1) were readily detectable by pyrosequencing-based analysis.When the proportions of mutant colorectal cancer cell line DNA were 5% and 10% content, the mutation rates of K-ras gene detected by conventional Sanger DNA sequencing were 33.3% (4/12) and 58.3% (7/12) respectively, whereas the mutation rates detected by pyrosequencing-based assay were 91.7% (11/12) and 100% (12/12) respectively, there were significant differences between those two sequencing methodology (P A transitions ［50%（5/10）］, followed by G>T transversions ［30%（3/10）］. Conclusion The pyrosequencing assay provides an accurate and sensitive method for mutation screening of K-ras exon 2 codons 12 and 13 in routine diagnostic specimens, thereby allowing the selection of the cancer treatment in clinical individualized practice.(Chin J Lab Med,2012,35:585-592) Key words: Colorectal neoplasms; Genes，ras; Exons; Mutation; Sequence analysis，DNA